Due in part to a number of new analytical techniques, there has been a significant increase in knowledge about genetic information, particularly in humans. Allelic variants of genetic loci have been correlated to malignant and non-malignant monogenic and multigenic diseases. For example, monogenic diseases for which the defective gene has been identified include DuChenne muscular dystrophy, sickle-cell anemia, Lesch Nyhan syndrome, hemophilia, beta-thalassemia, cystic fibrosis, polycystic kidney disease, ADA deficiency, .alpha.-1-antitrypsin deficiency, Wilm's tumor and retinoblastoma. Other diseases which are believed to be monogenic for which the gene has not been identified include fragile X mental retardation and Huntington's chorea.
Genes associated with multigenic diseases such as diabetes, colon cancer and premature coronary atherosclerosis have also been identified.
In addition to identifying individuals at risk for or carriers of genetic diseases, detection of allelic variants of a genetic locus has been used for organ transplantation, forensics, disputed paternity and a variety of other purposes in humans. In commercially important plants and animals, genes have not only been analyzed but genetically engineered and transmitted into other organisms.
A number of techniques have been employed to detect allelic variants of genetic loci including analysis of restriction fragment length polymorphic (RFLP) patterns, use of oligonucleotide probes, and DNA amplification methods. One of the most complicated groups of allelic variants, the major histocompatibility complex (MHC), has been extensively studied. The problems encountered in attempting to determine the HLA type of an individual are exemplary of problems encountered in characterizing other genetic loci.
The major histocompatibility complex is a cluster of genes that occupy a region on the short arm of chromosome 6. This complex, denoted the human leukocyte antigen (HLA) complex, includes at least 50 loci. For the purposes of HLA tissue typing, two main classes of loci are recognized. The Class I loci encode transplantation antigens and are designated A, B and C. The Class II loci (DRA, DRB, DQA1, DQB, DPA and DPB) encode products that control immune responsivenes. Of the Class II loci, all the loci are polymorphic with the exception of the DRA locus. That is, the DR.alpha. antigen polypeptide sequence is invariant.
HLA determinations are used in paternity determinations, transplant compatibility testing, forensics, blood component therapy, anthropological studies, and in disease association correlations to diagnose disease or predict disease susceptibility. Due power of HLA to distinguish individuals and the need to match HLA type for transplantation, analytical methods to unambiguously characterize the alleles of the genetic loci associated with the complex have been sought. At present, DNA typing using RFLP and oligonucleotide probes has been used to type Class II locus alleles. Alleles of Class I loci and Class II DR and DQ loci are typically determined by serological methods. The alleles of the Class II DP locus are determined by primed lymphocyte typing (PLT).
Each of the HLA analysis methods has drawbacks. Serological methods require standard sera that are not widely available and must be continuously replenished. Additionally, serotyping is based on the reaction of the HLA gene products in the sample with the antibodies in the reagent sera. The antibodies recognize the expression products of the HLA genes on the surface of nucleated cells. The determination of fetal HLA type by serological methods may be difficult due to lack of maturation of expression of the antigens in fetal blood cells.
Oligonucleotide probe typing can be performed in two days and has been further improved by the recent use of polymerase chain reaction (PCR) amplification. PCR-based oligoprobe typing has been performed on Class II loci. Primed lymphocyte typing requires 5 to 10 days to complete and involves cell culture with its difficulties and inherent variability.
RFLP analysis is time consuming, requiring about 5 to 7 days to complete. Analysis of the fragment patterns is complex. Additionally, the technique requires the use of labelled probes. The most commonly used label, .sup.32 P, presents well known drawbacks associated with the use of radionuclides.
A fast, reliable method of genetic locus analysis is highly desirable.